JP2012504964A - Compositions and methods for detecting antibodies specific for Anaplasma phagosite film (Aph) and Anaplasma platis (Apl) - Google Patents

Abstract

The present invention provides methods and compositions for the detection and treatment of Anaplasma phagosite film and Anaplasma platis infection.
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Description

Priority This application claims priority based on US Provisional Patent Application No. 61 / 103,743, filed Oct. 8, 2008, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION Anaplasmosis occurs in mammals (eg, humans, horses, sheep, dogs, cats, deer, and ruminants) and is a tick-borne pathogen, Anaplasma phagocytophilum (“Aph”). Caused by granulocyte cell infections (formerly known as Ehrlichia equi). Common clinical symptoms include fever, lethargy, difficulty walking, thrombocytopenia, lymphadenopathy, and anexoria, all of which are not specific for anaplasmosis. Therefore, specific tests are important for accurate diagnosis.

  Anaplasma platys ("Apl") (formerly known as Ehrlichia platys) is a related species. Apl causes canine infectious periodic thrombocytopenia (ICCT). Infected dogs are usually asymptomatic in the United States, but can be clinically ill in other parts of the world. Current Anaplasmic serological tests cannot distinguish between the two species due to significant cross-reactivity. There is a need in the art for methods of detecting Aph and ApI and methods for distinguishing between two infections.

  The onset of clinical symptoms occurs during the acute phase of anaplasmosis and can occur before the appearance of measurable levels of antibodies against some anaplasma antigens. Accordingly, there is a need for a rapid, sensitive, and reliable immunological test for Aph infection, Apl infection, or both (eg, in mammals exhibiting clinical symptoms of acute anaplasmosis).

SUMMARY OF THE INVENTION In one embodiment of the invention, it consists of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10, or amino acids 1-45 of SEQ ID NO: 10; amino acids 41 of SEQ ID NO: 10 -89; amino acids 85-130 of SEQ ID NO: 10; amino acids 126-160 of SEQ ID NO: 10; amino acids 129-146 of SEQ ID NO: 10; amino acids 144-160 of SEQ ID NO: 10; Consisting of amino acids 136-155 of NO: 10, or consisting of at least 17 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1-21, or at least 94% of SEQ ID NO: 1-21 Compositions containing one or more purified polypeptides consisting of polypeptides having identity are provided. The one or more purified polypeptides include an indicator, a signal sequence, a transport stop sequence, an amino acid spacer, a transmembrane domain, a protein purification ligand, a heterologous polypeptide, a moiety that enhances an immune response, a moiety that facilitates purification, It may be linked to a moiety that improves the stability of the peptide, one or more additional polypeptides containing SEQ ID NO: 1-21, or combinations thereof. In another aspect of the invention, an isolated polynucleotide that encodes one or more purified polypeptides is provided.

  In yet another aspect of the invention, a method of detecting an antibody that specifically binds to an Anaplasma phagosite film polypeptide or an Anaplasma platis polypeptide, or both, in a test sample is provided. The method includes contacting a composition containing one or more purified polypeptides with a test sample under conditions that allow formation of a polypeptide / antibody complex. The polypeptide / antibody complex is detected. Detection of the polypeptide / antibody complex indicates that antibodies specific for the Anaplasma phagosite film polypeptide or the Anaplasma platis polypeptide, or both, are present in the test sample. The composition containing one or more purified polypeptides may consist of at least 17 contiguous amino acids of the amino acid sequence of SEQ ID NO: 8 or 19. Detection of the polypeptide / antibody complex indicates that an antibody specific for Anaplasma platis is present in the test sample. The composition containing one or more purified polypeptides comprises an amino acid sequence of SEQ ID NO: 10; amino acids 41-89 of SEQ ID NO: 10; or at least 17 of amino acids 85-130 of SEQ ID NO: 10 A polypeptide comprising at least about 94% identity with SEQ ID NO: 10, amino acids 41-89 of SEQ ID NO: 10, or amino acids 85-130 of SEQ ID NO: 10 It may consist of Detection of the polypeptide / antibody complex indicates that antibodies specific for the Anaplasma phagosite film polypeptide or the Anaplasma platis polypeptide, or both, are present in the test sample. A composition containing one or more purified polypeptides comprises amino acids 1-45 of SEQ ID NO: 10; amino acids 126-160 of SEQ ID NO: 10; amino acids 129-146 of SEQ ID NO: 10; Consisting of at least 17 consecutive amino acids of amino acids 136-155 of SEQ ID NO: 10, or amino acids 1-45 of SEQ ID NO: 10; Amino acids 126-160; amino acids 129-146 of SEQ ID NO: 10; amino acids 144-160 of SEQ ID NO: 10; from polypeptides having at least about 94% identity to amino acids 136-155 of SEQ ID NO: 10 It may be made. Detection of the polypeptide / antibody complex indicates that an antibody specific for the Anaplasma phagosite film polypeptide is present in the test sample. Prior to detection, the complex may be contacted with an indicator. The amount of antibody in the test sample may be measured. One or more purified polypeptides may be bound to the substrate.

In yet another aspect of the invention, there are provided antibodies that specifically bind to a polypeptide consisting of SEQ ID NO: 1-21. The antibody may be a monoclonal antibody, a polyclonal antibody, a Fab fragment, a Fab ′ fragment, a Fab′-SH fragment, a F (ab ′) ₂ fragment, an Fv fragment, or a single chain antibody.

In yet another aspect of the invention, a method for detecting Anaplasma phagosite film polypeptide or Anaplasma platis polypeptide, or both in a sample is provided. The method comprises contacting the sample with one or more antibodies that specifically bind to one or more purified polypeptides of the invention under conditions that allow the formation of a polypeptide / antibody complex. Including. The polypeptide / antibody complex is detected. Detection of the polypeptide / antibody complex indicates that Anaplasma phagosite film polypeptide or Anaplasma platis polypeptide, or both, are present in the sample. One or more purified polypeptides are amino acids 1-45 of SEQ ID NO: 10; amino acids 126-160 of SEQ ID NO: 10; amino acids 129-146 of SEQ ID NO: 10; amino acids of SEQ ID NO: 10 144-160; or may consist of at least 17 consecutive amino acids of amino acids 136-155 of SEQ ID NO: 10. Detection of the polypeptide / antibody complex indicates that the Anaplasma phagosite film polypeptide is present in the test sample. One or more purified polypeptides may consist of at least 17 contiguous amino acids of the amino acid sequence of SEQ ID NO: 8 or 19. Detection of the polypeptide / antibody complex indicates that the Anaplasma platis polypeptide is present in the test sample. The one or more purified polypeptides comprise an amino acid sequence of SEQ ID NO: 10; amino acids 41-89 of SEQ ID NO: 10; or at least 17 consecutive amino acids of amino acids 85-130 of SEQ ID NO: 10 It may consist of Detection of the polypeptide / antibody complex indicates that Anaplasma phagosite film polypeptide or Anaplasma platis polypeptide is present in the test sample. The one or more antibodies may be monoclonal antibodies, polyclonal antibodies, Fab fragments, Fab ′ fragments, Fab′-SH fragments, F (ab ′) ₂ fragments, Fv fragments, or single chain antibodies.

  In another aspect of the invention, the composition containing the purified polypeptide of the invention may further contain a pharmaceutically acceptable or veterinary acceptable carrier and / or adjuvant.

  In yet another aspect of the invention, there is provided a method of treating or ameliorating Anaplasma platis infection, Anaplasma phagocytic film infection, or both in a mammalian subject, the method comprising one or more Administering a composition containing the purified polypeptide of the present invention to a mammalian subject in a therapeutically effective amount.

  In yet another aspect of the invention, a method for raising an immune response in a mammal is provided, the method comprising administering an immunologically effective amount of a composition comprising one or more purified polypeptides of the invention. Including doing.

  Thus, the present invention provides methods and compositions for the detection and treatment of ApI and / or Aph infections.

The results of assays performed using polypeptides containing SEQ ID NOs: 12-18 and 10 are shown. The results of assays performed using polypeptides containing SEQ ID NOs: 15, 19, and 10 are shown. The results of assays performed using polypeptides containing SEQ ID NOs: 15, 19, and 10 are shown. The results of a time course assay performed with polypeptides containing SEQ ID NOs: 15, 19, and 10 are shown. Figure 3 shows the results of a time course assay performed with polypeptides containing SEQ ID NOs: 15 and 10. Figure 3 shows the results of an assay performed with a polypeptide containing SEQ ID NO: 12-15. Figure 3 shows the results of an assay performed using a polypeptide containing SEQ ID NO: 20. Figure 3 shows the results of an assay performed using a polypeptide containing SEQ ID NO: 20.

Detailed Description of the Invention
Polypeptides of the Invention The singular forms ("a", "an", and "the") as used herein also include the plural forms unless otherwise indicated.

  A polypeptide is a polymer in which two or more amino acids are covalently linked by amide bonds. The polypeptide may be post-translationally modified. A purified polypeptide is a polypeptide preparation that is substantially free of cellular material, other types of polypeptides, chemical precursors, chemicals used to synthesize polypeptides, or combinations thereof. A polypeptide preparation that is substantially free of cellular material, media, chemical precursors, chemicals used to synthesize polypeptides, etc. may be used for other polypeptides, media, chemical precursors, and / or synthesis. Contains about 30%, 20%, 10%, 5%, 1% or less of other chemicals used. Thus, the purified polypeptide has a purity of about 70%, 80%, 90%, 95%, 99%, or more. Unpurified or semi-purified cell extracts or mixtures of polypeptides that are less than 70% pure are not included in the purified polypeptide.

  The term “polypeptide” may refer to one or more one type of polypeptide (a set of polypeptides). “Polypeptide” may also refer to a mixture of two or more different types of polypeptides (polypeptide mixture). The terms “polypeptide (s)” or “polypeptide (s)” may each mean “one or more polypeptides”.

In certain aspects of the invention, one or more of the following polypeptides are provided:

  In some embodiments of the invention, the X at position 143 of SEQ ID NO: 10 is K or N; the X at position 145 is T or A; and the X at position 156 is F or I.

In one aspect of the invention, the following polypeptides are provided:
1. Amino acids 1-45 of SEQ ID NO: 10 (Aph p44-1) (having the same reactivity as SEQ ID NO: 1 and 12)
2. Amino acids 41-89 (Aph p44-2) of SEQ ID NO: 10 (have the same reactivity as SEQ ID NO: 2 and 13)
3. Amino acids 85-130 (Aph p44-3) of SEQ ID NO: 10 (have the same reactivity as SEQ ID NO: 3 and 14)
4). Amino acids 126-160 (Aph p44-4) of SEQ ID NO: 10 (having the same reactivity as SEQ ID NOs: 4, 9, 11, 15, and 20)
5. Amino acids 129-146 of SEQ ID NO: 10 (Aph p44-4-1) (have the same reactivity as SEQ ID NOs: 5 and 16)
6). Amino acids 144-160 (Aph p44-4-2) of SEQ ID NO: 10 (having the same reactivity as SEQ ID NO: 6 and 17)
7). Amino acids 136-155 (Aph p44-3) of SEQ ID NO: 10 (having the same reactivity as SEQ ID NOs: 7 and 18)
Furthermore, SEQ ID NOs: 8 and 9 have the same reactivity, ie, their polypeptides bind substantially specifically to antibodies specific for Anaplasma antigen.

本発明のある態様では、SEQ ID NO:21の144位のXはKまたはNであり；146位のXはTまたはAであり；そして、157位のXはFまたはIである。 In certain embodiments of the invention, the polypeptide has a C-residue at the N-terminus. For example:

In certain embodiments of the invention, the X at position 144 of SEQ ID NO: 21 is K or N; the X at position 146 is T or A; and the X at position 157 is F or I.

SEQ ID NO: 4-7 and 9 can be aligned as follows:

本発明のある態様では、SEQ ID NO:11の18位のXはNまたはKであり；20位のXはTまたはAであり；そして、31位のXはFまたはIである。本発明のある態様は、SEQ ID NO:11のアミノ酸4-21；SEQ ID NO:11のアミノ酸19-34；またはSEQ ID NO:11のアミノ酸11-30を含む。 The consensus sequence of SEQ ID NO: 4-7 and 9 is shown in SEQ ID NO: 11:

In certain embodiments of the invention, the X at position 18 of SEQ ID NO: 11 is N or K; the X at position 20 is T or A; and the X at position 31 is F or I. Some embodiments of the invention include amino acids 4-21 of SEQ ID NO: 11; amino acids 19-34 of SEQ ID NO: 11; or amino acids 11-30 of SEQ ID NO: 11.

  In certain embodiments, about 46, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9 of SEQ ID NOs: 1-9 and 11-21 A purified polypeptide consisting of less than 8, 7, or 6 consecutive amino acids (or any number of amino acids between about 46 and about 6). In some embodiments, SEQ ID NOs: 1-9 and 11-12, about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 , 35, 40, or 46 or more consecutive amino acids (or any number of amino acids between about 46 and about 6). A natural Aph or Apl amino acid is any polypeptide naturally produced by Aph or Apl cells. A purified polypeptide can contain less than a certain number of contiguous Anaplasma amino acids of SEQ ID NO: 1-21 (eg, about 200, 175, 150, 125, 100, 75, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or less than 6 amino acids (or between about 200 and about 6) Any number of amino acids), i.e., purified polypeptides are smaller than full-length polypeptides, the fact that these polypeptides are smaller than full-length Anaplasma polypeptides is important, This is because, in Apl and / or Aph assays, smaller polypeptides can have higher specificity and / or sensitivity than full-length polypeptides, and these smaller polypeptides can be compared to full-length polypeptides. Manufacturing is cheaper and can be obtained with higher purity sell.

  In another embodiment, about 200, 175, 150, 125, 100, 75, 50, 25, 20, 15, 10, 6, or less contiguous naturally occurring Anaplasma amino acids of SEQ ID NO: 10 ( Or any number of amino acids between about 200 and about 6). In another embodiment, about 6, 10, 15, 25, 50, 75, 100, 125, 150, 175, 200, or more consecutive natural Anaplasma amino acids (or about A purified polypeptide consisting of any number between 6 and about 200 amino acids) is provided.

  At least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 with the polypeptide sequence shown in SEQ ID NO: 1-21 Variant polypeptides having 98, 99, or 99% identity are also polypeptides of the invention. For example, the variant polypeptide of SEQ ID NO: 1 has at least about 98% identity (about 1 amino acid change), 96% (about 2 amino acid change), 93% (about 3 amino acid change) with SEQ ID NO: 1 ), 91% (about 4 amino acid changes), 89% (about 5 amino acid changes), 87% (about 6 amino acid changes), 84% (about 7 amino acid changes), 82% (about 8 amino acid changes), or 80% (About 9 amino acid changes). The variant polypeptide of SEQ ID NO: 2 has at least about 98% identity (about 1 amino acid change), 96% (about 2 amino acid change), 94% (about 3 amino acid change) with SEQ ID NO: 2. 92% (about 4 amino acid changes), 90% (about 5 amino acid changes), 88% (about 6 amino acid changes), about 86% (about 7 amino acid changes), 84% (about 8 amino acid changes), 82% ( It may be about 9 amino acid changes), or 80% (about 10 amino acid changes). The variant polypeptide of SEQ ID NO: 3 has at least about 98% identity (about 1 amino acid change), 96% (about 2 amino acid change), 94% (about 3 amino acid change) with SEQ ID NO: 3 91% (about 4 amino acid changes), 89% (about 5 amino acid changes), 87% (about 6 amino acid changes), 85% (about 7 amino acid changes), 83% (about 8 amino acid changes), or 80% ( About 9 amino acid changes). The variant polypeptides of SEQ ID NOs: 4 and 9 have at least about 97% identity (about 1 amino acid change), 94% (about 2 amino acid change), 91% (about about 1 amino acid change) with SEQ ID NOs: 4 and 9. 3 amino acid changes), 89% (about 4 amino acid changes), 86% (about 5 amino acid changes), 83% (about 6 amino acid changes), or 80% (about 7 amino acid changes). The variant polypeptide of SEQ ID NO: 5 has at least about 94% (about 1 amino acid change), 89% (about 2 amino acid change), or 83% (about 3 amino acid change) identity to SEQ ID NO: 5 It may be. The variant polypeptide of SEQ ID NO: 6 is at least about 94% (about 1 amino acid change), 88% (about 2 amino acid change), or 82% (about 3 amino acid change) to SEQ ID NO: 6 It may be. The variant polypeptide of SEQ ID NO: 7 has at least about 95% identity (about 1 amino acid change), 90% (about 2 amino acid change), 85% (about 3 amino acid change) with SEQ ID NO: 7, Alternatively, it may be 80% (about 4 amino acid changes). The variant polypeptide of SEQ ID NO: 8 has at least about 97% identity (about 1 amino acid change), 94% (about 2 amino acid change), 91% (about 3 amino acid change) with SEQ ID NO: 8, It may be 88% (about 4 amino acid changes), 84% (about 5 amino acid changes), or 81% (about 6 amino acid changes).

  Variant polypeptides have one or more conservative amino acid mutations or other minor modifications and retain biological activity, i.e., biologically functional equivalents. Biologically active equivalents have substantially equivalent functions compared to the corresponding wild-type polypeptide. In certain embodiments of the invention, the polypeptide has about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or less conservative amino acid substitutions.

  Percent sequence identity has an art-recognized meaning and there are many ways to measure identity between two polypeptide or polynucleotide sequences. For example, see: Lesk, Computational Molecular Biology, Oxford University Press (New York), (1988); Smith, Biocomputing: Informatics And Genome Projects, Academic Press (New York), (1993); Griffin and Griffin, Computer Analysis Of Sequence Data, Part I, Humana Press (New Jersey), (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov and Devereux, Sequence Analysis Primer, M Stockton Press ( New York), (1991). Polynucleotide or polypeptide alignment methods have been organized into computer programs, such as the GCG program package (Devereux et al., Nuc. Acids Res. 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J Molec. Biol. 215: 403 (1990)) and the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) (Smith and Waterman (Adv App. Math., 2: 482-489 (1981))))). For example, the computer program ALIGN using the FASTA algorithm may be used with an affine gap search with a gap open penalty = -12 and a gap extension penalty = -2.

  When using any sequence alignment program to determine whether a particular sequence is, for example, about 95% identical to a reference sequence, the percent identity is calculated over the entire length of the reference polynucleotide and the identity The parameters are set so that the gap is allowed up to 5% of the total number of nucleotides in the reference polynucleotide.

  In general, the identification of a mutant polypeptide involves modifying one of the polypeptide sequences of the invention and evaluating the properties of the modified polypeptide to determine whether it is a biological equivalent. Can be done by. A variant reacts substantially similarly to a polypeptide of the invention in an assay (eg, immunohistochemical assay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoenzyme assay, or Western blot assay). If so (eg, having 90-110% activity of the original polypeptide), the variant is a bioequivalent. In certain embodiments, the assay is a competitive assay and the biologically equivalent polypeptide is capable of reducing the binding of a polypeptide of the invention to the corresponding reactive antigen or antibody by about 80, 95, 99, or 100%. Have An antibody that specifically binds to the corresponding wild-type polypeptide also specifically binds to the mutant polypeptide.

  A conservative substitution is one in which one amino acid is replaced with another with similar properties and is not expected to substantially alter the polypeptide's secondary structure and hydropathy by those skilled in the art of peptide chemistry. Is. In general, the following amino acid groups show conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

  The polypeptides of the present invention may further contain a signal (or leader) sequence that directs transport of the protein simultaneously with or after translation. A polypeptide may also contain linkers or other sequences to facilitate synthesis, purification, or identification of the polypeptide (eg, poly-His), or to facilitate binding of the polypeptide to a solid support. Good. For example, the polypeptide may be conjugated to an immunoglobulin Fc region or bovine serum albumin.

  A polypeptide may be covalently or non-covalently bound to an amino acid sequence that is not normally associated in nature, ie, a heterologous amino acid sequence. The heterologous amino acid sequence may be a non-Anaplasma phagosite film organism, a non-Anaplasma platis cell, a synthetic sequence, or an Anaplasma phagosite film that is not usually located at the carboxy or amino terminus of the polypeptide of the invention It may be derived from an array or an Anaplasma platis array. Furthermore, the polypeptide may be covalently or non-covalently bound to compounds or molecules other than amino acids (eg, indicators). The polypeptide may be covalently or non-covalently linked to an amino acid spacer, amino acid linker, signal sequence, transport stop sequence, transmembrane domain, protein purification ligand, or combinations thereof. A polypeptide also has a moiety that enhances the immune response (ie, a functional group (which may be a polypeptide or other compound)) (eg, a cytokine such as IL-2), a moiety that facilitates purification (eg, 6- Histidine tags, trpE, glutathione, affinity tags such as maltose binding protein), or moieties that improve polypeptide stability (eg, polyethylene glycol; amino-terminal protecting groups (eg, acetyl, propyl, succinyl, benzyl, benzyloxy) Carbonyl, or t-butyloxycarbonyl); carboxyl-terminal protecting groups such as amide, methylamide, and ethylamide)). In certain embodiments of the invention, the protein purification ligand may be one or more C amino acid residues (eg, located at the amino terminus or carboxy terminus of a polypeptide of the invention). An amino acid spacer is an amino acid sequence that is not naturally associated with a polypeptide of the invention. The amino acid spacer may contain about 1, 5, 10, 20, 100, or 1,000 amino acids.

  If desired, the polypeptides of the present invention may be part of a fusion protein that contains other amino acid sequences (eg, amino acid linkers, amino acid spacers, signal sequences, TMR transport stop sequences, transmembrane domains), and It may further contain ligands useful for protein purification (eg glutathione-S-transferase, histidine tag, and staphylococcal protein A), or combinations thereof. Other amino acid sequences may be present at the C or N terminus of the polypeptides of the invention to form fusion proteins. More than one polypeptide of the invention may be present in the fusion protein. Fragments of the polypeptides of the invention may be present in the fusion proteins of the invention. A fusion protein of the invention may contain one or more polypeptides of the invention, fragments thereof, or combinations thereof.

  The polypeptide of the present invention may be a multimer. That is, the polypeptide may contain one or more copies of the polypeptide of the invention, or a combination thereof. The multimeric polypeptide may be a multi-antigen peptide (MAP). See, for example, Tam, J. Immunol. Methods, 196: 17-32 (1996).

  The polypeptides of the present invention may also contain antigenic determinants recognized by antibodies specific for Anaplasma phagosite film or Anaplasma platis, or for both Anaplasma phagosite film and Anaplasma platis. Good. A polypeptide may contain one or more epitopes (ie antigenic determinants). The epitope may be a linear epitope, a sequential epitope, or a conformational epitope. Epitopes in the polypeptides of the invention can be identified by several methods. See, for example, U.S. Pat. No. 4,554,101; Jameson and Wolf, CABIOS 4: 181-186 (1988). For example, the polypeptides of the present invention may be isolated and screened. A series of short peptides that together cover the entire polypeptide sequence may be prepared by proteolytic cleavage. For example, starting from a 30 amino acid long polypeptide fragment, each fragment may be tested for the presence of an epitope recognized by ELISA. For example, in an ELISA assay, an Anaplasma phagosite film polypeptide (eg, a 30 amino acid long polypeptide fragment) is bound to a solid support (eg, a well of a plastic multiwell plate). A group of antibodies are labeled and added to a solid support, allowed to bind to unlabeled antigen under conditions that inhibit non-specific adsorption, and unbound antibodies and other proteins are washed away. Antibody binding is detected, for example, by a reaction that turns a colorless substrate into a colored reaction product. Thereafter, the overlapping fragments that are gradually smaller from the identified 30 amino acid length may be examined and mapped to the epitope of interest.

  The polypeptide of the present invention may be produced recombinantly. The polynucleotide encoding the polypeptide of the present invention may be introduced into a recombinant expression vector using techniques known in the art and expressed in a suitable expression host cell system. A variety of bacterial, yeast, plant, mammalian, and insect expression systems are available in the art, any of which may be used. If necessary, the polynucleotide encoding the polypeptide may be translated in a cell-free translation system. The polypeptide may be chemically synthesized or obtained from Anaplasma cells.

  The immunogenic polypeptides of the present invention may contain the amino acid sequence shown in SEQ ID NO: 1-21 or a fragment thereof. An immunogenic polypeptide can elicit an antibody or other immune response (eg, a T cell response of the immune system) that recognizes an epitope of a polypeptide having SEQ ID NO: 1-21. The immunogenic polypeptide of the present invention may be a fragment of a polypeptide having the amino acid sequence shown in SEQ ID NO: 1-21. The immunogenic polypeptide fragments of the present invention are about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, It may be 60, 70, 80, 90, 100, 125, 150, 175, 200, or more amino acids long. The immunogenic polypeptide fragments of the present invention are about 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, It may be 14, 13, 12, 11, 10, 9, 8, 7, 6, or less amino acids long.

Anaplasma Polynucleotides The polynucleotides of the present invention may contain less than the entire microbial genome, and may be single-stranded or double-stranded nucleic acids. The polynucleotide may be RNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA, or a combination thereof. The polynucleotide may be purified so that it does not contain other components (eg, proteins, lipids, and other polynucleotides). For example, the polynucleotide may be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% pure. For example, nucleic acid molecules present in hundreds to millions of other nucleic acid molecules in a cDNA or genomic library, or gel slices containing genomic DNA restriction enzyme digests are not considered purified polynucleotides. The polynucleotide of the present invention encodes the above-described polypeptide of the present invention. In certain embodiments of the invention, the polynucleotide encodes the polypeptide set forth in SEQ ID NO: 1-21 or a fragment thereof.

  The polynucleotides of the present invention comprise about 639, 450, 300, 225, 147, 138, 135, 105, 96, 60, 54, 51, 45, 30, 20, 10, or less contiguous natural forms (ie Aph Or an ApI polynucleotide) or a non-naturally occurring polynucleotide. The polynucleotides of the present invention comprise about 10, 20, 30, 45, 51, 54, 60, 96, 105, 135, 138, 147, 225, 300, 450, 639, or more contiguous natural forms (ie, Aph). Or an ApI polynucleotide) or a non-naturally occurring polynucleotide. A purified polynucleotide may contain additional heterologous nucleotides (ie, nucleotides that are not derived from Aph or Apl) and additional Aph or Apl nucleotides that are not naturally contiguous to the polynucleotide. The polynucleotides of the present invention may comprise other nucleotide sequences (eg, linkers, signal sequences, TMR transport stop sequences, transmembrane domains, or ligands useful for protein purification (eg, glutathione-S-transferase, histidine tag, and staphylococcal protein). It may contain a sequence encoding A).

  The polynucleotide of the present invention may be isolated. An isolated polynucleotide is a naturally occurring polynucleotide that is not directly adjacent to one or both of the 5 'and 3' flanking genomic sequences that are naturally associated. For example, an isolated polynucleotide may be a recombinant DNA molecule of any length, except that the nucleic acid sequence immediately adjacent to the recombinant DNA molecule in the naturally occurring genome has been removed. Or does not exist. Isolated polynucleotides also include non-natural nucleic acid molecules.

  The polynucleotide of the present invention may comprise a fragment encoding an immunogenic polypeptide. The polynucleotides of the invention may encode full-length polypeptides, polypeptide fragments, and mutant or fusion polypeptides.

  A degenerate nucleotide sequence encoding a polypeptide of the invention, as well as at least about 80%, or about 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 with the polynucleotide sequence of the invention. Homologous nucleotide sequences having% identity and their complements are also polynucleotides of the invention. The percent sequence identity can be calculated as described in the section “Polypeptides”. A degenerate nucleotide sequence is a polynucleotide that encodes a polypeptide of the invention or a fragment thereof, but whose nucleic acid sequence differs from the wild-type polynucleotide sequence due to the degeneracy of the genetic code. Complementary DNA (cDNA) molecules, species homologs, and variants of the polynucleotides of the invention that encode biologically functional polypeptides are also polynucleotides of the invention.

  The polynucleotide of the present invention may be obtained from a nucleic acid sequence present in, for example, a biological sample (such as blood, serum, saliva, or tissue from an infected individual). Polynucleotides may be synthesized in the laboratory using, for example, an automated synthesizer. Amplification methods such as PCR may be used to amplify the polynucleotide from either genomic DNA or cDNA encoding the polypeptide.

  The polynucleotides of the invention may contain a coding sequence for a naturally occurring polypeptide or may encode a modified sequence that does not occur in nature. If necessary, the polynucleotide may be cloned into an expression vector containing an expression control element (for example, an origin of replication, promoter, enhancer, or other control element) that causes expression of the polynucleotide of the present invention in a host cell. The expression vector may be, for example, a plasmid (such as pBR322, pUC, or ColE1) or an adenovirus vector (an adenovirus type 2 vector or a type 5 vector). Other vectors may be used if desired, including but not limited to: Sindbis virus, Simian virus 40, alphavirus vector, poxvirus vector, cytomegalovirus and retro Viral vectors such as mouse sarcoma virus, mouse mammary tumor virus, Moloney murine leukemia virus, and rous sarcoma virus. Minichromosomes (eg MC and MC1), bacteriophages, phagemids, yeast artificial chromosomes, bacterial artificial chromosomes, viral particles, virus-like particles, cosmids (plasmids into which the cos site of λ phage has been inserted), and replicons (in cells, Genetic elements that can replicate under their own control) may be used.

  The preparation of polynucleotides operably linked to expression control sequences and methods for their expression in host cells are well known in the art. See, for example, US Pat. No. 4,366,246. A polynucleotide of the invention is operably linked if it is adjacent to or located in the vicinity of one or more expression control elements that direct transcription and / or translation of the polynucleotide. .

  The polynucleotide of the present invention may be used, for example, as a probe or primer (eg, a PCR primer) to detect the presence of an anaplasma polynucleotide in a test sample (eg, a biological sample). A probe is a molecule that has the ability to interact with a target nucleic acid, typically in a sequence-specific manner, for example by hybridization. Primers are a subset of probes that can assist in enzyme treatment and can hybridize with a target nucleic acid to cause enzyme treatment. Primers may be prepared from any combination of nucleotides or nucleotide derivatives or analogs that can be used in the art and that do not interfere with enzyme treatment.

  The probe or primer is about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, which encodes a polypeptide of the invention. There may be 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or more contiguous nucleotides.

  Nucleic acid hybridization is well understood in the art and is also described herein. In general, probes may be prepared from any combination of nucleotides or nucleotide derivatives or analogs that can be used in the art. The ability of these probes and primers to specifically hybridize to Anaplasma phagosite film or Anaplasma platis polynucleotide sequences is used to detect the presence of complementary sequences in a given test sample be able to. The polynucleotide probes and primers of the invention are complementary sequences in a test sample, eg, a biological sample (eg, saliva, sputum, blood, plasma, serum, urine, feces, cerebrospinal fluid, amniotic fluid, wound exudate, or tissue). Can be hybridized. The sample-derived polynucleotide may be subjected to, for example, gel electrophoresis or other size separation methods, or may be immobilized without size separation. The polynucleotide probe or primer may be labeled. Suitable labels and methods for labeling probes and primers are known in the art and include, for example, radioactive labels introduced by nick translation or kinase, biotin labels, fluorescent labels, chemiluminescent labels, bioluminescent labels , Metal chelate labels, and enzyme labels. The polynucleotide from the sample is contacted with a probe or primer under suitable stringency hybridization conditions.

  Depending on the application, various hybridization conditions can be used to vary the degree of selectivity of the probe or primer relative to the target sequence. In applications where a high degree of selectivity is required, relatively high stringency conditions, such as low salt concentrations and / or high temperature conditions (eg, about 50 ° C. to about 70 ° C. at a salt concentration of about 0.02M to about 0.15M). May be used). For applications that require less selectivity, lower stringency conditions can be used. For example, a temperature of about 20 ° C. to about 55 ° C. at a salt concentration of about 0.14M to about 0.9M. The presence of a hybridized complex containing a probe or primer and a complementary polynucleotide from the test sample indicates that an ApI or ApI polynucleotide sequence is present in the sample.

Antibody The antibody of the present invention is an antibody molecule that specifically binds to an Anaplasma phagosite film polypeptide, an Anaplasma platis polypeptide, a mutant polypeptide of the present invention, or a fragment thereof. The antibodies of the invention may be specific for Aph polypeptides, Apl polypeptides, variant polypeptides, or combinations thereof, eg, antibodies specific for one or more of SEQ ID NO: 1-21 It may be. In another embodiment of the invention, the antibody is specific for an Anaplasma phagosite film polypeptide (eg, SEQ ID NOs: 1, 4, 5, 6, 7, 9, 11, 12, 15, 16, Antibodies specific for 17, 18, or 20). In another embodiment of the invention, the antibody is specific for an Anaplasma platis polypeptide (eg, an antibody specific for SEQ ID NO: 8 or 19). In another embodiment of the invention, the antibody is specific for both an Anaplasma phagocyte film polypeptide and an Anaplasma platis polypeptide (eg, SEQ ID NO: 2, 3, 10, 13, 14, Or 21 specific antibodies). One of ordinary skill in the art can readily determine whether an antibody is specific for an Anaplasma phagosite film polypeptide or an Anaplasma platis polypeptide using the assays described herein. The antibodies of the present invention may be polyclonal antibodies, monoclonal antibodies, single chain antibodies (scFv), or antigen-binding fragments of antibodies. An antigen-binding fragment of an antibody is a portion of an intact antibody that contains the antigen-binding site or variable region of the intact antibody, and that portion does not contain the constant heavy chain domain of the Fc region of the intact antibody. Examples of antigen-binding antibody fragments include Fab, Fab ′, Fab′-SH, F (ab ′) ₂ , and _Fv fragments.

  The antibodies of the invention may be of any antibody class, such as IgG, IgM, IgA, IgD, and IgE. The antibody or fragment thereof binds to an epitope of the polypeptide of the invention. Antibodies may be made in vivo in suitable laboratory animals or in vitro by recombinant DNA techniques. Antibody preparation and characterization methods are well known in the art. For example, see: Dean, Methods Mol. Biol. 80: 23-37 (1998); Dean, Methods Mol. Biol. 32: 361-79 (1994); Baileg, Methods Mol. Biol. 32: 381- 88 (1994); Gullick, Methods Mol. Biol. 32: 389-99 (1994); Drenckhahn et al. Methods Cell. Biol. 37: 7-56 (1993); Morrison, Ann. Rev. Immunol. 10: 239-65 (1992); Wright et al. Crit. Rev. Immunol. 12: 125-68 (1992). For example, polyclonal antibodies are prepared by administering a polypeptide of the invention to an animal (eg, a human or other primate, mouse, rat, rabbit, guinea pig, goat, pig, dog, cow, sheep, donkey, or horse). May be. Serum from the immunized animal is collected and the antibody is purified from plasma by, for example, ammonium sulfate precipitation followed by chromatography (eg, affinity chromatography). Polyclonal antibody preparation and processing methods are known in the art.

“Specifically binds” or “specific for” means that the first antigen (eg, Anaplasma phagosite film or Anaplasma platis polypeptide) binds the antibody of the present invention to other non-specific It recognizes and binds with higher affinity for the target molecule. A non-specific molecule is an antigen that does not share a common epitope with the first antigen. In a preferred embodiment of the invention, the nonspecific molecule is not derived from an Anaplasma species. The Anaplasma species is any species of the Anaplasma genus. For example, an antibody raised against a first antigen (eg, a polypeptide) that binds with higher efficiency than a non-specific antigen may be expressed as specifically binding to the first antigen. it can. In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to the polypeptide of SEQ ID NO: 1-21 or a fragment thereof when binding with a binding affinity of K _a = 10 ⁷ l / mol or greater. Specific binding can be tested by methodology well known in the art using, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay.

The antibodies of the present invention include the following antibodies and antigen-binding fragments thereof:
(A) competes with a reference antibody for binding to SEQ ID NO: 1-21 or antigen-binding fragment thereof; (b) binds to the same epitope of SEQ ID NO: 1-21 or antigen-binding fragment thereof as the reference antibody. (C) binds to SEQ ID NO: 1-21 or an antigen-binding fragment thereof with substantially the same K _d as the reference antibody; and / or (d) SEQ ID NO at substantially the same off-rate as the reference antibody. NO: 1-21 or is (here coupled to antigen-binding fragment thereof, the reference antibody is SEQ ID NO: 1-21 of the polypeptide or K _a = 10 ⁷ l / mol or higher binding affinity to the antigen binding fragments Or an antigen-binding fragment thereof that specifically binds at a).

  Furthermore, a monoclonal antibody that targets an epitope present on the polypeptide of the present invention can also be easily produced. For example, normal B cells derived from a mammal (eg, a mouse) immunized with the polypeptide of the present invention may be fused with, for example, HAT-sensitive mouse myeloma cells to produce a hybridoma. Hybridomas producing Anaplasma specific antibodies may be identified using RIA or ELISA and isolated by cloning on semi-flow agar or limiting dilution. Clones producing Anaplasma specific antibodies are isolated by further screening. Screening for monoclonal antibody specificity may be performed by standard methods, such as by binding a polypeptide of the invention to a microtiter plate and measuring monoclonal antibody binding by an ELISA assay. Methods for producing and processing monoclonal antibodies are known in the art. See, for example, Kohler and Milstein, Nature, 256: 495 (1975). Monoclonal antibodies of a particular isotype can be prepared directly from the initial fusion, or can be isolated from class hybrids using sib selection methods from parental hybridomas that secrete monoclonal antibodies of different isotypes. It may be prepared secondarily by releasing. See Steplewski et al., P.N.A.S. U.S.A. 82: 8653 1985; Spria et al., J. Immunolog. Meth. 74: 307, 1984. The monoclonal antibody of the present invention may be a recombinant monoclonal antibody. See, for example, US Pat. No. 4,474,893; US Pat. No. 4,816,567. The antibody of the present invention may be chemically constructed. See, for example, US Pat. No. 4,676,980.

  The antibodies of the invention can be chimeric antibodies (see, eg, US Pat. No. 5,482,856), humanized antibodies (eg, Jones et al., Nature 321: 522 (1986); Reichmann et al., Nature 332: 323 (1988); Presta, Curr. Op. Struct. Biol. 2: 593 (1992)), canineized antibodies, canine antibodies, or human antibodies. Human antibodies may be generated, for example, by direct immortalization, phage display, transgenic mice, or the Trimera method (see, eg, Reisener et al., Trends Biotechnol. 16: 242-246 (1998)).

  Specific for Anaplasma phagosite film antigen (eg, SEQ ID NO: 1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, or 20) but not Anaplasma platis antigen The antibody that binds to the presence of Anaplasma phagosite film antigen in a sample (eg, a serum, blood, plasma, urine, stool, tissue, cell, or saliva sample from an animal infected with Anaplasma phagocytosis film) It is particularly useful for the detection of

  An antibody that specifically binds to an Anaplasma platis antigen (eg, SEQ ID NO: 8 or 19) but not an Anaplasma phagocytosis film antigen is a sample (eg, serum, blood from animals infected with Anaplasma platis, blood, It is particularly useful for detecting the presence of Anaplasma platis antigens in plasma, urine, stool, tissue, or saliva samples.

  Antibodies that specifically bind to Anaplasma platis and Anaplasma phagosite film antigen (eg, SEQ ID NO: 2, 3, 10, 13, 14, or 21) have been infected with a sample (eg, Anaplasma platis) Serum, blood, plasma, urine, stool, tissue, or saliva samples from animals or animals infected with Anaplasma phagosite film) for the detection of the presence of Anaplasma platis antigen and Anaplasma phagosite film antigen It is particularly useful.

  Immunoassays for Anaplasma antigens may use one antibody or multiple antibodies. An immunoassay for an anaplasma antigen is, for example, a monoclonal antibody (s) specific for an anaplasma epitope (s), a combination of monoclonal antibodies (s) specific for an anaplasma polypeptide epitope (s), Monoclonal antibodies specific to epitopes of different Anaplasma polypeptides (multiple), polyclonal antibodies specific to the same Anaplasma antigen (s), specific to different Anaplasma antigens Polyclonal antibody (s), or a combination of monoclonal and polyclonal antibodies may be used. The immunoassay protocol may be based on, for example, competition, direct reaction, or a sandwich type assay (eg, using a labeled antibody). The antibodies of the present invention may be labeled with any type of label known in the art (eg, fluorescence, chemiluminescence, radioactivity, enzyme, colloidal metal, radioisotope, and bioluminescent label). The antibody of the present invention may specifically bind to Aph antigen alone, Apl antigen alone, or Aph antigen and Apl antigen.

  The antibody or fragment thereof of the present invention may be bound to a support and used to detect the presence of ApI and / or Aph antigen. Supports include, for example, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite.

  Furthermore, the antibodies of the present invention may be used to isolate ApI and / or Aph cells or antigens by immunoaffinity columns. The antibody may be immobilized on a solid support such that it retains its immunologically selective activity (eg, by adsorption or covalent bonding). If necessary, a spacer group may be contained so as to keep the antigen-binding site of the antibody accessible. Then, using the immobilized antibody, Anaplasma cells or Anaana derived from a sample (for example, biological sample (saliva, serum, sputum, blood, urine, feces, cerebrospinal fluid, amniotic fluid, wound exudate, or tissue)) Plasma antigen may be bound. The bound Anaplasma cells or Anaplasma antigen is recovered from the column matrix, for example by pH change.

  The antibodies of the invention may also be used in immunolocalization studies to analyze the presence and distribution of the polypeptides of the invention during various cellular events or physiological conditions. Furthermore, antibodies may be used to identify molecules involved in passive immunity and molecules involved in non-protein antigen biosynthesis. Identification of these molecules can be useful in vaccine development. The antibodies of the present invention (eg, monoclonal antibodies and single chain antibodies) may be used to monitor the course of remission of diseases caused by ApI and / or Aph. By measuring the increase or decrease of ApI and / or Aph specific antibodies in an animal derived test sample, it may be determined whether a particular treatment regimen aimed at ameliorating the disease is effective. Antibody detection and / or quantification may be performed, for example, using direct binding assays such as RIA, ELISA, or Western blot assays.

Detection Methods Using the methods of the present invention, specific to Anaplasma antigen, Apl antigen, Aph antigen, Apl polynucleotide, Aph polynucleotide, or combinations thereof in a test sample, eg, a biological sample, environmental sample, or laboratory sample Antibodies or specific binding fragments thereof may be detected. The test sample is an Apl polynucleotide, Aph polynucleotide, Apl polypeptide, Anaplasma species polypeptide, Aph polypeptide, an antibody specific for Anaplasma species, an antibody specific for Apl, and / or Aph specific It may contain an antibody, an irrelevant polynucleotide, polypeptide, antibody, or antigen, or a combination of the above, or may not contain any of the above. Biological samples include, for example, serum, blood, cells, plasma, saliva, urine, stool, or tissue from a mammal (such as a horse, cat, dog, or human). The test sample can be untreated or subjected to precipitation, fractionation, separation, dilution, concentration, or purification.

  In certain embodiments, the methods of the invention comprise contacting one or more polypeptides of the invention with a test sample under conditions that allow formation of a polypeptide / antibody complex (ie, an immune complex). Including. That is, the polypeptide of the present invention specifically binds to an antibody specific for ApI and / or Aph antigen present in a sample. In certain embodiments of the invention, one or more polypeptides of the invention (eg, SEQ ID NOs: 1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, 20, Or a fragment thereof) specifically binds to an antibody that is specific for the Aph antigen and does not specifically bind to the Apl antigen. In another embodiment of the invention, one or more polypeptides of the invention (eg, SEQ ID NO: 2, 3, 10, 13, 14, 21, or fragments thereof) are specific for both Aph and Apl antigens. Specifically bind to a specific antibody. In certain embodiments of the invention, one or more polypeptides of the invention (eg, SEQ ID NO: 8, 19, or a fragment thereof) are specific for Apl antigen and do not specifically bind to Aph antigen. It binds specifically to the antibody. Those skilled in the art are familiar with the assays and conditions used to detect antibody / polypeptide complex binding. Complex formation between the polypeptide and anti-Apl and / or anti-Aph antibody in the sample is detected. The formation of an antibody / polypeptide complex indicates the presence of Anaplasma phagosite film polypeptide and / or Anaplasma platis polypeptide in the sample. If no polypeptide / antibody complex is detected, this indicates that there is no Anaplasma phagosite film polypeptide and / or Anaplasma platis polypeptide in the sample.

  The antibodies of the invention can be used in a method of diagnosing ApI and / or Aph infection, for example by taking a test sample from a human or animal suspected of being infected with ApI and / or Aph. A test sample is contacted with an antibody of the invention under conditions that allow the formation of an antibody-antigen complex (ie, an immune complex). Conditions suitable for the formation of antigen / antibody complexes are known to those skilled in the art. The amount of the antibody-antigen complex may be measured by a method known in the art. A level higher than that generated in the control sample indicates infection with ApI and / or Aph. A control sample is a sample that does not contain any Aph and / or Apl polypeptide or Aph and / or Apl specific antibodies. In certain embodiments of the invention, the control does not contain an Anaplasma species polypeptide or an Anaplasma species specific antibody. In certain embodiments of the invention, the antibody is specific only for the Aph antigen. In another embodiment of the invention, the antibody is specific for both Aph and Apl antigens. In another embodiment of the invention, the antibody is specific only for the ApI antigen. Alternatively, the polypeptide of the present invention may be contacted with a test sample. Antibodies specific for ApI and / or Aph in a positive test sample form an antigen-antibody complex under suitable conditions. The amount of the antibody-antigen complex may be measured by a method known in the art.

  In certain embodiments of the invention, Anaplasma phagosite film and / or Anaplasma platis infection may be detected in a subject. A biological sample is taken from the subject. One or more of the purified SEQ ID NO: 1-21-containing polypeptides or other polypeptides of the invention is contacted with a biological sample under conditions that allow formation of a polypeptide / antibody complex. The polypeptide / antibody complex is detected. Detection of the polypeptide / antibody complex indicates that the mammal is infected with Anaplasma phagosite film and / or Anaplasma platis. If no polypeptide / antibody complex is detected, it indicates that the mammal is not infected with Anaplasma phagosite film or Anaplasma platis.

  Since SEQ ID NO: 2, 3, 10, 13, 14, and 21 are specific for both anti-Apl and anti-Aph antibodies, the detected infection is Aph infection, Apl infection, or both Apl and Aph May be an infection. SEQ ID NO: 1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, and 20 are specific for anti-Aph antibodies, so the detected infection is an Aph infection . Since SEQ ID NO: 8 and 19 are specific for anti-Apl antibodies, the detected infection is ApI infection. If no polypeptide / antibody complex is detected, the subject is not infected with Anaplasma phagosite film or Anaplasma platis.

  In some embodiments of the invention, the ApI and / or Aph infection is about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days after the subject's Apl and / or Aph infection, Detected in the subject by 13, 14, 15, 16, 17, 18, 19, 20, 21, 21 or later. In certain embodiments of the invention, the ApI and / or Aph infection is about 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days after the subject's Apl and / or Aph infection, Detected in the subject after 13, 12, 11, 10, 9, 8, 7, 6, 5, or earlier.

  In certain embodiments of the invention, a polypeptide / antibody complex is detected when an indicator bound to the antibody (eg, an enzyme conjugate) catalyzes a detectable reaction. If desired, an indicator containing a signal generating compound may be applied to the polypeptide / antibody complex under conditions that allow formation of the polypeptide / antibody / indicator complex. The polypeptide / antibody / indicator complex is detected. Optionally, the polypeptide / antibody complex is formed after labeling the polypeptide or antibody with an indicator. The method may optionally include a positive or negative control.

  In certain embodiments of the invention, one or more antibodies of the invention are bound to a solid phase or substrate. A test sample that may contain a protein comprising a polypeptide of the invention is added to the substrate. One or more antibodies that specifically bind to the polypeptides of the invention are added. The antibody can be the same as the antibody used on the solid phase, or can be from a different source or species, and can be conjugated to an indicator (eg, an enzyme conjugate). A washing step may be performed before each addition. Add chromophore or enzyme substrate and allow to color. The color reaction is stopped and the color is quantified using, for example, a spectrophotometer.

  In another aspect of the invention, one or more antibodies of the invention are bound to a solid phase or substrate. A test sample that may contain a protein comprising a polypeptide of the invention is added to the substrate. A second anti-species antibody that specifically binds to the polypeptide of the present invention is added. These second antibodies are from a different species than the solid phase antibodies. A third anti-species antibody that specifically binds to the second antibody and does not specifically bind to the solid phase antibody is added. The third antibody may contain an indicator (eg, an enzyme conjugate). A washing step may be performed before each addition. Add chromophore or enzyme substrate and allow to color. The color reaction is stopped and the color is quantified using, for example, a spectrophotometer.

  Assays of the present invention include, but are not limited to, those based on competition, direct reaction, or sandwich-type assays, including but not limited to enzyme-linked immunosorbent assays (ELISA), Western There are blots, IFA, radioimmunoassay (RIA), hemagglutination (HA), fluorescence polarization immunoassay (FPIA), and microtiter plate assays (assays performed in one or more wells of a microtiter plate). Certain assays of the invention include reversible flow chromatography binding assays, such as the SNAP® assay. See, for example, US Pat. No. 5,726,010.

  The assay may be performed using a solid phase or substrate, or by any other method that does not use immunoprecipitation or a solid phase. When using a solid phase or substrate, one or more polypeptides of the present invention are bound directly or indirectly to a solid support or substrate such as: microtiter wells, magnetic beads, Fibrous mats, particulate materials (non-magnetic beads, columns, matrices, membranes, synthetic or natural fibers (for example, materials based on glass or cellulose, or thermoplastic polymers such as polyethylene, polypropylene, or polyester) For example, a sintered structure made of glass or various thermoplastic polymers) or a cast film made of nitrocellulose, nylon, polysulfone, etc. (generally synthetic in nature). In one embodiment of the present invention, the substrate is sintered fine polyethylene particles, commonly known as porous polyethylene (eg, 10-15 micron porous polyethylene manufactured by Chromex, Albuquerque, New Mexico). ). All of these substrate materials can be used in a suitable shape (eg film, sheet or plate) or can be coated on a suitable inert carrier (eg paper, glass, plastic film or fabric) Or it may be bonded or laminated to it. Suitable methods for immobilizing peptides on the solid phase include ionic interactions, hydrophobic interactions, covalent interactions and the like.

  In some assay formats, one or more polypeptides may be coated on a solid phase or substrate. A test sample suspected of containing an anti-Apl and / or anti-Aph antibody or antigen-binding fragment thereof, together with an indicator comprising a signal-generating compound conjugated to an antibody or antigen-binding antibody fragment specific for Apl and / or Aph The antigen / antibody complex is bound by either the test sample antibody binding to the solid phase polypeptide, or an indicator conjugated to an antibody specific for Apl and / or Aph binding to the solid phase polypeptide. Incubate for a time and under conditions sufficient to form. The reduction in binding of the indicator conjugated to the anti-Apl and / or anti-Aph antibody to the solid phase may be quantitatively measured. If there is a measurable decrease in signal compared to the signal generated from a test sample that has been confirmed to be Apl negative and / or Aph negative, this is an anti-Apl and / or anti-Aph antibody in the test sample. Indicates that exists. This type of assay can quantify anti-Apl and / or anti-Aph antibodies in a test sample.

  In another type of assay format, one or more polypeptides of the invention are coated on a support or substrate. A polypeptide of the invention is conjugated to an indicator and added to the test sample. This mixture is applied to a support or substrate. If antibodies specific for Apl and / or Aph are present in the test sample, they are one or more of the polypeptides conjugated to the indicator and one of the polypeptides immobilized on the support or Join more than that. This polypeptide / antibody / indicator complex can then be detected. In this type of assay, the amount of anti-Apl and / or anti-Aph antibody in the test sample can be quantified.

  In another type of assay format, one or more polypeptides of the invention are coated on a support or substrate. A test sample is applied to a support or substrate and incubated. The solid support is washed with a washing solution to remove unbound components derived from the sample. If antibodies specific for Apl and / or Aph are present in the test sample, they bind to the polypeptide coated on the solid phase. This polypeptide / antibody complex can be detected using a second species-specific antibody conjugated to an indicator. The polypeptide / antibody / anti-species antibody indicator complex can then be detected. In this type of assay, the amount of anti-Apl and / or anti-Aph antibody in the test sample can be quantified.

  Formation of the polypeptide / antibody complex or polypeptide / antibody / indicator complex can be detected, for example, by radiometry, colorimetry, fluorescence measurement, size separation, or precipitation. Optionally, the polypeptide / antibody complex is detected by adding a secondary antibody conjugated to an indicator containing a signal generating compound. Indicators comprising a signal generating compound (label) bound to a polypeptide / antibody complex can be detected by the methods described above, including chromophores, catalysts (eg enzyme conjugates), fluorescent compounds (eg fluorescein and rhodamine), There are chemiluminescent compounds (eg dioxetane, acridinium, phenanthridinium, ruthenium, and luminol), radioactive elements, directly visible labels, and cofactors, inhibitors, magnetic particles, and the like. Examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase, β-galactosidase and the like. The choice of a particular label is not critical, but is capable of generating a signal by itself or conjugated with one or more additional substances.

  Complex formation indicates the presence of anti-Apl and / or anti-Aph antibodies in the test sample. Thus, the methods of the present invention can be used to diagnose ApI and / or Aph infections in patients.

  The method of the invention can also indicate the amount of anti-Apl and / or anti-Aph antibody in the test sample. With many indicators (eg enzyme conjugates), the amount of antibody present is proportional to the signal produced. Depending on the type of test sample, it can be diluted with a suitable buffer, concentrated, or contacted with a solid phase without any manipulation. For example, it is usually preferred to test a pre-diluted serum or plasma sample, or concentrated specimen (eg, urine) to determine the presence and / or amount of antibody.

  The invention further includes assay kits (eg, products) that detect anti-Apl and / or anti-Aph antibodies or antibody fragments, Apl polypeptides, and / or Aph polypeptides in a sample. The kit includes one or more polypeptides of the invention and means for determining the binding of the polypeptide to an anti-Apl antibody and / or anti-Aph antibody or antigen-binding antibody fragment in a sample. The kit or product also determines the binding of the antibody or antigen-binding antibody fragment to one or more of the antibodies or antigen-binding antibody fragments of the invention and the Apl polypeptide and / or Aph polypeptide in the sample. Means may be included. A kit comprises a device containing one or more polypeptides or antibodies of the invention, and one or more polypeptides or antibodies (eg, for identification of ApI and / or Aph infections in mammals). Instructions for use may be included. The kit may also include packaging material that includes a label indicating that one or more polypeptides or antibodies of the kit can be used to identify ApI and / or Aph infections. Other components known to those skilled in the art (eg, buffers, controls, etc.) may be included in these test kits. The polypeptides, antibodies, assays, and kits of the present invention are useful, for example, in the diagnosis of individual cases of ApI and / or Aph infections in patients and epidemiological studies of the spread of ApI and / or Aph.

  The polypeptides and assays of the present invention may be used in conjunction with other polypeptides or assays to detect the presence of Anaplasma along with other organisms. For example, the polypeptides and assays of the invention may be used in conjunction with reagents that detect canine filamentous worms and / or Borrelia burgdorferi and / or Ehrlichia canis.

  The polynucleotides of the invention can be used to detect the presence of ApI and / or Aph polynucleotides in a sample. Polynucleotides can be used to detect ApI and / or Aph polynucleotides in a sample by a simple hybridization reaction, which can also be used, for example, in a polymerase chain reaction (PCR) (eg, a real-time PCR reaction). it can. The methods and compositions of the present invention can also be used to distinguish and detect the presence of Aph from other Anaplasma species (eg, ApI).

  PCR assays are known in the art and are described, for example, in US Pat. No. 4,683,195; US Pat. No. 4,683,202; US Pat. No. 4,965,188. In general, a polynucleotide primer is annealed to a denatured strand of the target nucleic acid. Polymerization of deoxynucleoside triphosphate with polymerase generates a primer extension product. PCR then involves repeated cycles of template nucleic acid denaturation, primer annealing, and annealing primer extension by the action of a thermostable polymerase. This step results in exponential amplification of the target Anaplasma species nucleic acid in the test sample, allowing detection of target polynucleotides present at very low concentrations in the sample.

  Real-time PCR assays are based on the detection of a signal (eg a fluorescent reporter signal). This signal increases in direct proportion to the amount of PCR product in the reaction. Real-time PCR is an amplification method that makes it possible to monitor the progress of an ongoing amplification reaction. See Quantitation of DNA / RNA Using Real-Time PCR Detection, Perkin Elmer Applied Biosystems (1999); PCR Protocols (Academic Press New York, 1989). By recording the amount of fluorescence emitted at each cycle, the PCR reaction in the exponential amplification phase can be monitored (where the initial significant increase in the amount of PCR product correlates with the initial amount of target template) ). The higher the starting nucleic acid target copy number, the earlier a significant increase in fluorescence is observed.

  Certain embodiments of the invention provide methods for detecting and / or quantifying ApI and / or Aph polynucleotides in a test sample. Sense and antisense primers may be added to the test sample under conditions suitable for the polymerase chain reaction. If an ApI and / or Aph polynucleotide is present in the test sample, the primer hybridizes with the ApI and / or Aph polynucleotide to form an amplification product. Amplification products are detected and the presence and / or amount of ApI and / or Aph polynucleotides is determined. Detection of amplification products can be performed using polynucleotide probes that hybridize with ApI and / or Aph polynucleotide sequences under conditions suitable for polymerase chain reaction. The amplification product may be quantified by measuring the detection signal from the probe and comparing the detection signal with a second probe detection signal from a quantification standard. The quantitative standard may be extracted in parallel with the test sample.

In one aspect of the invention, a method is provided for distinguishing and detecting Anaplasma phagosite film polypeptides in a sample from Anaplasma platis polypeptides. The method includes:
(A) one or more antibodies that specifically bind to a polypeptide consisting of SEQ ID NO: 1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, or 20; In contact with the sample under conditions allowing the formation of a polypeptide / antibody complex and detecting the polypeptide / antibody complex; and (b) specifically for a polypeptide consisting of SEQ ID NO: 8 or 19 Contacting the sample with one or more antibodies that bind to the sample under conditions that allow the formation of the polypeptide / antibody complex, and detecting the polypeptide / antibody complex.

  If a polypeptide / antibody complex is detected in steps (a) and (b), the sample contains an Anaplasma phagosite film polypeptide and contains an Anaplasma platis polypeptide. If the polypeptide / antibody complex is detected in step (a) and not detected in step (b), the sample contains an Anaplasma phagosite film polypeptide, but does not contain an Anaplasma platis polypeptide do not do. If the polypeptide / antibody complex is not detected in step (a) and is detected in step (b), the sample contains an Anaplasma platis polypeptide, but the Anaplasma phagosite film polypeptide is Does not contain. If no polypeptide complex is detected in step (a) and not detected in step (b), the sample does not contain an Anaplasma platis polypeptide and does not contain an Anaplasma phagosite film polypeptide. .

In another aspect of the present invention, an Anaplasma platis polypeptide, an Anaplasma phagosite film polypeptide, or both an Anaplasma platis polypeptide and an Anaplasma phagosite film polypeptide are specific. Methods are provided for detecting antibodies that bind. The method includes:
(A) one or more purified polypeptides containing SEQ ID NO: 1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, or 20 polypeptide / antibody Contacting the test sample under conditions allowing the formation of a complex and detecting the polypeptide / antibody complex; and (b) one or more purified polypeptides comprising SEQ ID NO: 8 or 19 Is contacted with a test sample under conditions that allow formation of the polypeptide / antibody complex, and the polypeptide / antibody complex is detected.

  If a polypeptide / antibody complex is detected in step (a) and step (b), the sample is an antibody that specifically binds to the Anaplasma phagosite film polypeptide and the Anaplasma platis polypeptide (ie , An antibody having the ability to specifically bind to both the Anaplasma platis polypeptide and the Anaplasma phagocyte film polypeptide). If a polypeptide / antibody complex is detected in step (a) and not detected in step (b), the sample contains antibodies that specifically bind to the Anaplasma phagosite film polypeptide, but Anaplasma Does not contain antibodies that specifically bind to Platis polypeptides. If the polypeptide / antibody complex is not detected in step (a) and is detected in step (b), the sample contains an antibody that specifically binds to the Anaplasma platis polypeptide, It does not contain antibodies that specifically bind to phagocytic film polypeptides. If the polypeptide complex is not detected in step (a) and not detected in step (b), the sample does not contain an antibody specific for Anaplasma platis polypeptide, It also does not contain antibodies specific for the polypeptide.

Methods for treating, ameliorating, or preventing diseases caused by Aph and / or Apl Using the polypeptides, polynucleotides, and antibodies of the present invention, treating, ameliorating diseases caused by Apl and / or Aph, Or it can be prevented. For example, an antibody (eg, a monoclonal antibody of the invention or an antigen-binding fragment thereof) may be administered to an animal (eg, a human or dog). In certain embodiments of the invention, the antibody or antigen-binding fragment thereof is administered to an animal as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutical composition comprises a therapeutically effective amount of the antibody or antigen-binding fragment thereof. A therapeutically effective amount is an amount effective to ameliorate symptoms of ApI and / or Aph infection or reduce the amount of ApI and / or Aph cells in a subject.

  The polypeptide or polynucleotide of the present invention may be included in an immunogenic composition and used to elicit an immune response in a host. An immunogenic composition or immunogen has the ability to induce an immune response in an animal. The immunogenic polypeptides or polynucleotide compositions of the present invention are particularly useful for sensitizing an animal's immune system and consequently eliciting an immune response that ameliorates or prevents the effects of ApI and / or Aph infections. . Eliciting an immune response in an animal model can be useful, for example, in determining the optimal dose or route of administration. Eliciting an immune response may be used to treat, prevent, or ameliorate a disease or infection caused by ApI and / or Aph. The immune response includes a humoral immune response or a cellular immune response, or a combination thereof. The immune response may include the promotion of the host's systemic response, for example by promoting the production of defensins.

  In certain aspects of the invention, immunogens comprising the polypeptides of the invention and one or more additional regions or moieties covalently linked to the carboxyl terminus or amino terminus of the polypeptide are provided. Each region or portion can, for example, enhance an immune response, facilitate purification of the immunogen, or improve the stability of the polypeptide.

  Production of antibody titers against ApI and / or Aph by animals can be important for protection from infection and clearance of infection. Detection and / or quantification of antibody titers after polypeptide or polynucleotide delivery can be used to identify epitopes that are particularly effective in inducing antibody titers. Identification of epitopes involved in strong antibody responses to Apl and / or Aph can be performed by raising antibodies against various lengths of Apl and / or Aph polypeptides. The antibodies elicited by a particular polypeptide epitope can then be tested, for example using an ELISA assay, to determine which polypeptide contains the most effective epitope for eliciting a strong response. Thereafter, polypeptides or fusion proteins comprising those epitopes or polynucleotides encoding the epitopes may be constructed and used to elicit strong antibody responses.

  The polypeptide, polynucleotide, or antibody of the present invention is a mammal (eg, mouse, rabbit, guinea pig, macaque, baboon, chimpanzee, human, cow, sheep, pig, horse, dog, cat), or a chicken or duck It may be administered to animals to elicit antibodies in vivo. Polynucleotide injection is practically beneficial because it facilitates construction and modification. Furthermore, the polypeptide is synthesized in the host by injection of the polynucleotide. Thus, the polypeptide is presented to the host immune system with natural post-translational modifications, structure, and conformation. The polynucleotide may be delivered to the subject as “naked DNA”.

  Administration of the polynucleotide, polypeptide, or antibody may be by any method known in the art, including intramuscular, intravenous, intrapulmonary, intramuscular, intradermal, intraperitoneal, or subcutaneous injection, There are aerosols, intranasal, infusion pumps, suppositories, mucous membranes, topical, and oral, for example, injections using living ballistic guns (“gene guns”). The polynucleotide, polypeptide, or antibody may be accompanied by a protein carrier for oral administration. A combination of administration methods may be used to elicit an immune response. The antibody may be administered at a daily dose of about 0.5 mg to about 200 mg. In certain embodiments of the invention, the antibody is administered at a daily dose of about 20 to about 100 mg.

  Pharmaceutically acceptable carriers and diluents for use in therapy, and veterinary acceptable carriers and diluents are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing (AR Gennaro ed. 1985)). The carrier must not induce the production of antibodies that are themselves harmful to the host. These carriers include, but are not limited to: large, slowly metabolized macromolecules such as proteins, polysaccharides (latex functional SEPHAROSE®, agarose, cellulose, cellulose beads, etc.), polylactic acid , Polyglycolic acid, amino acid polymers (eg, polyglutamic acid, polylysine, etc.), amino acid copolymers, peptoids, lipitoids, and inactive non-pathogenic viral particles or bacterial cells. Liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesives may be used as carriers for the compositions of the present invention.

  For example, the following pharmaceutically acceptable salts may be used in the compositions of the present invention: inorganic salts such as hydrochloride, hydrobromide, phosphate, or sulfate, and organic acid salts , For example acetate, proprionates, malonate or benzoate. Particularly useful protein substrates are serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those skilled in the art. The compositions of the present invention may be liquids or excipients such as water, saline, phosphate buffered saline, Ringer's solution, Hank's solution, glucose, glycerol, dextrose, malodextrin, ethanol, etc. (alone or in combination), and You may contain substances, such as a wetting agent, an emulsifier, an osmotic pressure regulator, surfactant, or a pH buffer. Additional activators, such as bactericides, may be used.

Optionally, costimulatory molecules that improve immunogenic presentation to lymphocytes, such as B7-1 or B7-2, or cytokines such as MIP1α, GM-CSF, IL-2, and IL-12 are compositions of the invention You may make it contain. If necessary, an adjuvant may be contained in the composition. An adjuvant is a substance that can be used to nonspecifically enhance a specific immune response. Generally, the adjuvant and the polypeptide of the present invention are mixed and presented to the immune system or presented separately but at the same site in the animal. Adjuvants include, for example, oily adjuvants (eg, Freund's complete and incomplete adjuvants), inorganic salts (eg, Alk (SO ₄ ) ₂ , AlNa (SO ₄ ) ₂ , AlNH ₄ (SO ₄ ), silica, alum, Al (OH) ₃ , and Ca ₃ (PO ₄ ) ₂ ), polynucleotides (ie poly IC and poly AU acids), and certain natural substances such as wax D from Mycobacterium tuberculosis, and Corynebacterium parvum ( Corynebacterium parvum), Bordetella pertussis, and substances found in Brucella members. Adjuvants that can be used include, but are not limited to: MF59-0, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl -Nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, also known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'- Dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, also known as MTP-PE), and RIBI (three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton ( MPL + TDM + CWS) containing 2% squalene / TWEEN® 80 (polysorbate) emulsion).

  The composition of the present invention may be dispensed and taken as tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, injections, mouthwashes, dentrifices and the like. The percentage of one or more of the polypeptides, polynucleotides or antibodies of the present invention in their compositions and formulations varies from 0.1% to 60% of the unit weight.

  Administration of the polypeptide, polynucleotide, or antibody elicits an immune response in the animal that lasts at least 1 week, 1 month, 3 months, 6 months, 1 year, or longer. If necessary, animals may be boosted by one or more boosters of polypeptide, polynucleotide, or antibody 1 month, 3 months, 6 months, 1 year, or later after the initial injection. The immune response in may be maintained. If desired, costimulatory molecules or adjuvants may be provided before, after, or with the composition.

  To elicit an immune response such that a composition of the invention comprising a polypeptide, polynucleotide, antibody, or combination thereof is detected (eg, by ELISA) in a manner that is compatible with the particular composition used. Administer in an effective amount. The polynucleotide is administered to a mammal (eg, baboon, chimpanzee, dog, or human) at 1 ng / kg, 10 ng / kg, 100 ng / kg, 1000 ng / kg, 0.001 mg / kg, 0.1 mg / kg, or 0.5 mg / kg. It may be injected intramuscularly at a dose. The polypeptide or antibody may be injected intramuscularly into the mammal at a dose of 0.01, 0.05, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5, or 10 mg / kg.

  Polypeptides, polynucleotides, or antibodies, or combinations thereof may be administered to animals that are not infected with ApI and / or Aph, or may be administered to animals infected with ApI and / or Aph. An immunologically effective amount or therapeutically effective amount means that administration of the amount to an individual (either as a single dose or as part of a continuous dose) is effective in treating, ameliorating or preventing ApI and / or Aph infections. It means that there is. The particular dose of polynucleotide, polypeptide, or antibody in the composition depends on many factors, including but not limited to the species, age, and sex of the mammal to which the composition is administered. There are modes of administration of concomitant drugs, systemic symptoms, and compositions. An effective amount of the composition of the invention can be readily determined using only routine experimentation.

  All patents, patent applications, and other scientific or technical literature cited herein are hereby incorporated by reference in their entirety. The invention is described herein by way of example, as appropriate, with any element (s) or limitation (s) (which are not specifically disclosed herein). You may carry out in the state which lacked. Thus, for example, in each example described herein, the terms “comprising”, “consisting essentially of”, and “consisting of” may each be replaced with either of the other two terms. Well, it still retains its normal meaning. The terms and phrases used are intended to be illustrative rather than restrictive, and are intended to exclude any equivalents or parts of features shown and described in their use. However, it will be appreciated that various modifications are possible within the scope of the present invention. Accordingly, although the present invention has been specifically disclosed according to aspects and features as necessary, those skilled in the art may implement modifications and changes to the concept disclosed in the present specification. It should be understood that it is considered to be within the scope of the present invention as defined in the following claims.

  Further, when describing features or aspects of the present invention in Markush groups or other options, the present invention thereby recognizes individual members or subgroups of members of the Markush group or other groups, as will be appreciated by those skilled in the art. But it is also described.

Examples Example 1 Detection of Anti-Aph and Anti-Apl Antibodies Using Polypeptide Derived from Aph p44 Protein Polypeptides as shown in SEQ ID NOs: 12-18 and 10 (0.5 μg mixed in carbonate buffer (pH 9.6)) Immulon® 4 microtiter plates were coated overnight at In all examples, the polypeptide shown in SEQ ID NO: 10 has an N at position 143, an A at position 145, and an I at position 156.

The plate was washed twice with PetChek® wash buffer. The plate was blocked with 0.1 M Tris (pH 7.6) containing 2% TWEEN® 20 (polysorbate) /2.5% sucrose for 2 hours, and then dried overnight using a desiccant. Test samples diluted 1: 200 with conjugate diluent were added to the plates and incubated for 40 minutes. The plate was washed 6 times with PetChek® wash buffer. HRP conjugate rabbit anti-dog IgG (H + L) diluted 1: 2000 with conjugate diluent (Jackson ImmunoResearch Laboratories, West Grove, Pa .; catalog number: 304-035-003) was added to the plate, Incubated for 40 minutes. The plate was washed 6 times with PetChek® wash buffer. 60 μl of 3,3 ′, 5,5′-tetramethylbenzidine (“TMB”) was added to the plate and incubated for 10 minutes. 50 μl of stop solution was added and A650 was measured. The test samples are as follows:
APL_13DPI: Dog experimentally infected with ApI (13 days after infection)
APL_78DPI: Dog experimentally infected with ApI (78 days after infection)
APH: Pool of samples from Aph-infected dogs (7 specimens) from Minnesota
neg: Randomly extracted pool of healthy dog (7 samples) samples

The results are shown in Table 1 and FIG.

  All eight tested peptides tested positive for reactivity with a serum pool from Aph-infected dogs (7 samples) from Minnesota. rp44, p44-2, and p44-3 showed cross-reactivity with the serum of dogs experimentally infected with ApI at two different time points of infection. The reactivity of experimentally infected ApI-infected dog sera with p44-1, p44-4, p44-4-1, p44-4-2, and p44-4-3 was almost at the background level. It was. Thus, the polypeptides p44-1, p44-4, p44-4-1, p44-4-2, and p44-4-3 are not cross-reactive with sera from Apl-infected dogs.

Example 2 Species-specific detection of Aph or Apl in field samples from endemic areas Polypeptide mixed with carbonate buffer (pH 9.6) (Aph p44-4 and Apl p44-4 are 0.5 μg / mL; Aph rp44 is Imulon® 4 plates were coated overnight at 0.25 μg / mL). The plate was washed twice with PetChek® wash buffer and then blocked with 0.1 M Tris (pH 7.6) containing 2% TWEEN® 20 (polysorbate) /2.5% sucrose for 2 hours. Plates were dried overnight with desiccant. 25 μL of the test sample is 50 μL of peptide: HRP conjugate (p44-4-Aph (SEQ ID NO: 15): 0.5 μg / mL for HRP conjugate, p44-4-Apl (SEQ ID NO: 19): HRP conjugate 1 μg / mL for the gate and 3 μg / mL for the Aph rp44 conjugate) and incubated on the microtiter plate (incubation time is 1 hour for the experiment shown in FIG. 2 and 1 hour for the experiment shown in FIG. 3 45 Minutes). The plate was washed 6 times with PetChek® wash buffer. 60 μL of TMB was added to the plate and incubated for 10 minutes. 50 μL of stop solution was added and A650 was measured. Cut-off values were determined based on reactivity with 10 negative samples; cut-off = mean + 2 x SD (standard deviation).

  FIG. 2 shows the results of using Anaplasma Platis positive samples derived from dogs living in Anaplasma Platis epidemic areas (HP: Arizona, P: Bahamas). rp44 (SEQ ID NO: 10) was positive in 4 of 5 “HP” samples and positive in 7 of 7 “P” samples. Aph p44-4 (SEQ ID NO: 15) was positive in 0 of 5 “HP” samples and positive in 7 “P” samples. Apl p44-4 (SEQ ID NO: 19) was positive in 5 “HP” samples and positive in 7 “P” samples. Therefore, Aph p44-4 does not cross react with sera from dogs infected with Apl.

  FIG. 3 shows the results of using an Anaplasma phagosite film positive sample derived from a dog living in an Aph endemic area (ME: Minnesota). rp44 (SEQ ID NO: 10) showed strong positive results in 21 of 22 ME samples. Aph p44-4 (SEQ ID NO: 15) showed strong positive results in 20 of the 22 “ME” samples. Apl p44-4 (SEQ ID NO: 19) showed negative results in 18 of 22 samples and very weak positive results in 4 of 22 ME samples. Therefore, Appl44-4 is considered not to cross-react with sera from dogs infected with Aph.

Example 3 Time course response in Apl experimental infection model Immulon with polypeptide mixed in carbonate buffer (pH 9.6) (Aph p44-4 and Apl p44-4 0.5 μg / mL; Aph rp44 0.25 μg / mL) 4 plates were coated overnight. The plate was washed 3 times with PetChek® wash buffer. The plate was blocked with 0.1 M Tris (pH 7.6) containing 2% TWEEN® 20 (polysorbate) /2.5% sucrose for 2 hours and left to dry overnight. Mix 25 μL test sample with 50 μL peptide: HRP conjugate (0.5 μg / mL for Aph p44-4 conjugate, 1 μg / mL for Apl p44-4 conjugate, 3 μg / mL for Aph rp44 conjugate) and micro Incubate for 1 hour on titer plate. The plate was washed 6 times with PetChek® wash buffer. 60 μL of TMB was added and incubated for 10 minutes. 50 μL of stop solution was added and A650 was measured. The results are shown in FIG.

  The results show that SEQ ID NO: 19 (Apl p44-4) can be used to detect ApI infection. SEQ ID NO: 19 detects ApI infection about 10 days after infection. Aph p44-4 (SEQ ID NO: 15) does not show cross-reactivity with sera from dogs infected with ApI. Aph rp44 (SEQ ID NO: 10) cross-reacts with sera from Apl-infected dogs in about 14 days and can be used to detect Apl and Aph infections.

Example 4 Response over time in Aph experimental infection model
Aph p44-4 (SEQ ID NO: 15) and Aph rp44 (SEQ ID NO: 10) were tested at a series of time points for reactivity with sera from dogs experimentally infected with Aph. Eleven randomly selected healthy field dog samples were also tested. Immulon® 4 plates were coated overnight with a polypeptide (Aph p44-4 0.5 μg / mL; Aph rp44 0.25 μg / mL) mixed in carbonate buffer (pH 9.6). The plate was washed 4 times with PetChek® wash buffer. The plate was blocked with 0.1% Tris (pH 7.6) containing 2% TWEEN® 20 (polysorbate) /2.5% sucrose for 2 hours, and the plate was dried overnight using a desiccant. 25 μL of test sample is mixed with 50 μL of peptide: HRP conjugate (p44-4-Aph (SEQ ID NO: 15): 0.5 μg / mL for HRP conjugate, 3 μg / mL for Aph rp44 conjugate) Added. Plates were incubated for 1 hour. The plate was washed 6 times with PetChek® wash buffer. TMB was added to the plate and incubated for 10 minutes. 50 μL of stop solution was added and A650 was measured. The results are shown in FIG. Aph p44-4 (SEQ ID NO: 15) was able to detect Aph infection about 10-14 days after infection. Aph rp44 (SEQ ID NO: 10) was able to detect Aph infection about 10 days after infection.

Example 5 Detection of Acute Aph Infection Immulon® 4 plates were coated overnight with polypeptide (0.5 μg / mL or 1.0 μg / mL) mixed in carbonate buffer (pH 9.6). The plate was washed 4 times with PetChek® wash buffer and then blocked with 0.1 M Tris (pH 7.6) containing 2% TWEEN® 20 (polysorbate) /2.5% sucrose for 2 hours. Plates were dried overnight with desiccant. 100 μl of serum test sample diluted 1: 200 with sample diluent was incubated for 45 minutes. The plate was washed 6 times with PetChek® wash buffer. Add 100 μL of HRP-conjugated rabbit anti-dog IgG (H + L) diluted 1: 2000 with sample diluent (Jackson ImmunoResearch Laboratories, West Grove, PA; catalog number: 304-035-003) for 45 minutes Incubated. The plate was washed 6 times with PetChek® wash buffer. 60 μl of TMB was added to the plate and incubated for 10 minutes. 50 μl of stop solution was added and A650 was measured. The test samples are as follows:
Positive (“+”) indicates a pool of 7 serum samples from late Aph-infected dogs.
Negative (“-”) indicates a pool of 7 healthy canine serum samples randomly extracted.
VML8, VML14, VML21, and VML156 represent serum samples from 4 dogs that are Aph positive for IFA and show acute clinical signs of anaplasmosis. The results are shown in FIG. Aph p44-4 reacted with 4 sera. Aph p44-1 and Aph p44-2 did not react with 4 sera, and Aph p44-3 reacted slightly with 4 sera. Thus, Aph p44-4 and Aph 44-3 can be used for the detection of Aph in subjects exhibiting acute clinical signs of anaplasmosis.

Example 6 Peptide p44-4-v performance
Aph p44-4-v (SEQ ID NO: 20) was tested using the samples shown in Table 2.

  Immulon® 4 plates were coated overnight with polypeptide (0.5 μg / mL) mixed in carbonate buffer (pH 9.6). The plate was washed twice with PetChek® wash buffer and then blocked with 0.1 M Tris (pH 7.6) containing 2% TWEEN® 20 (polysorbate) /2.5% sucrose for 2 hours. Plates were dried overnight with desiccant. Test samples diluted 1: 200 with conjugate diluent were added to the plates and incubated for 40 minutes. The plate was washed 6 times with PetChek® wash buffer. HRP conjugate rabbit anti-dog IgG (H + L) diluted 1: 2000 with conjugate diluent (Jackson ImmunoResearch Laboratories, West Grove, Pa .; catalog number: 304-035-003) was added to the plate, Incubated for 40 minutes. The plate was washed 6 times with PetChek® wash buffer. 60 μl of TMB was added to the plate and incubated for 10 minutes. 50 μl of stop solution was added and A650 was measured. The results are shown in FIG. 7, which shows the average value of duplicate experiments. In p44-4-v, the following samples showed positive results: VML21; ILS73, APH, and + ve; the following samples showed negative results: APL and -ve. Therefore, Aph 44-4-v can detect Aph infection in acute cases in a short period of at least 14 days after infection. Aph p44-4-v is not cross-reactive with sera from Apl-infected dogs.

Claims (22)

  Consisting of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10; or amino acids 1-45 of SEQ ID NO: 10; amino acids 41-89 of SEQ ID NO: 10; amino acids 85- of SEQ ID NO: 10 130; amino acids 126-160 of SEQ ID NO: 10; amino acids 129-146 of SEQ ID NO: 10; amino acids 144-160 of SEQ ID NO: 10; or amino acids 136-155 of SEQ ID NO: 10; or A purified polypeptide comprising at least 17 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1-21; or a purified polypeptide comprising a polypeptide having at least about 94% identity to SEQ ID NO: 1-21 A composition containing one or more.   2. An isolated polynucleotide encoding one or more purified polypeptides of claim 1.   One or more of the purified polypeptides is an indicator, a signal sequence, a transport stop sequence, an amino acid spacer, a transmembrane domain, a protein purification ligand, a heterologous polypeptide, a portion that enhances an immune response, a portion that facilitates purification, 2. The composition of claim 1 conjugated to a moiety that improves stability, one or more additional polypeptides comprising SEQ ID NO: 1-21, or combinations thereof. A method for detecting antibodies that specifically bind to Anaplasma phagosite film polypeptide or Anaplasma platis polypeptide, or both, in a test sample, the following:
(A) contacting a composition containing one or more purified polypeptides of claim 1 with a test sample under conditions that allow formation of a polypeptide / antibody complex;
(B) detecting a polypeptide / antibody complex;
Wherein the detection of the polypeptide / antibody complex indicates the presence of an antibody specific for the Anaplasma phagosite film polypeptide or the Anaplasma platis polypeptide, or both, in the test sample.   One or more of the purified polypeptides is an indicator, a signal sequence, a transport stop sequence, a transmembrane domain, an amino acid spacer, a protein purification ligand, a heterologous polypeptide, a portion that enhances an immune response, a portion that facilitates purification, 5. The composition of claim 4 conjugated to a moiety that improves stability, one or more additional polypeptides comprising SEQ ID NO: 1-21, or combinations thereof.   A composition containing one or more purified polypeptides consists of at least 17 contiguous amino acids of the amino acid sequence of SEQ ID NO: 8 or 19, and detection of the polypeptide / antibody complex is performed in the test sample. 5. The method of claim 4, wherein the method indicates the presence of an antibody specific for plasma platis.   A composition containing one or more purified polypeptides comprises the amino acid sequence of SEQ ID NO: 10; amino acids 41-89 of SEQ ID NO: 10; or at least 17 of amino acids 85-130 of SEQ ID NO: 10 Or from a polypeptide having at least about 94% identity with SEQ ID NO: 10; amino acids 41-89 of SEQ ID NO: 10; or amino acids 85-130 of SEQ ID NO: 10 5. The detection of polypeptide / antibody complex indicates the presence of an antibody specific for Anaplasma phagosite film polypeptide or Anaplasma platis polypeptide, or both, in the test sample. Method.   A composition containing one or more purified polypeptides is SEQ ID NO: 10 amino acids 1-45, SEQ ID NO: 10 amino acids 126-160, SEQ ID NO: 10 amino acids 129-146; SEQ ID NO: 10 Consisting of at least 17 consecutive amino acids of amino acids 144-160 of NO: 10, amino acids 136-155 of SEQ ID NO: 10; or amino acids 1-45 of SEQ ID NO: 10, amino acids of SEQ ID NO: 10 126-160, amino acids 129-146 of SEQ ID NO: 10; consisting of a polypeptide having at least about 94% identity to amino acids 144-160 of SEQ ID NO: 10, amino acids 136-155 of SEQ ID NO: 10 5. The method of claim 4, wherein the detection of the polypeptide / antibody complex indicates the presence of an antibody specific for the Anaplasma phagosite film polypeptide in the test sample.   5. The method of claim 4, further comprising contacting the complex of step (a) with an indicator prior to performing step (b).   The method of claim 4, wherein the amount of antibody in the test sample is measured.   5. The method of claim 4, wherein one or more purified polypeptides are bound to the substrate.   An antibody that specifically binds to a polypeptide consisting of SEQ ID NO: 1-21. 13. The antibody of claim 12, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a Fab fragment, a Fab ′ fragment, a Fab′-SH fragment, a F (ab ′) ₂ fragment, an Fv fragment, or a single chain antibody. A method for detecting Anaplasma phagosite film polypeptide or Anaplasma platis polypeptide or both in a sample, including:
(A) contacting one or more antibodies that specifically bind to one or more purified polypeptides of claim 1 with a sample under conditions that allow formation of a polypeptide / antibody complex; thing;
(B) detecting a polypeptide / antibody complex;
Wherein the detection of the polypeptide / antibody complex indicates the presence of Anaplasma phagosite film polypeptide or Anaplasma platis polypeptide, or both, in the sample.   One or more purified polypeptides are amino acids 1-45 of SEQ ID NO: 10, amino acids 126-160 of SEQ ID NO: 10, amino acids 129-146 of SEQ ID NO: 10, amino acids of SEQ ID NO: 10 144-160, consisting of at least 17 contiguous amino acids of amino acids 136-155 of SEQ ID NO: 10, and detection of the polypeptide / antibody complex indicates the presence of an Anaplasma phagosite film polypeptide in the test sample The method of claim 14.   The one or more purified polypeptides consist of at least 17 contiguous amino acids of the amino acid sequence of SEQ ID NO: 8 or 19, and detection of the polypeptide / antibody complex is an Anaplasma platis polypeptide in the test sample The method of claim 14, wherein the method indicates the presence of   One or more purified polypeptides from the amino acid sequence of SEQ ID NO: 10; amino acids 41-89 of SEQ ID NO: 10; or from at least 17 consecutive amino acids of amino acids 85-130 of SEQ ID NO: 10 15. The method of claim 14, wherein detection of the polypeptide / antibody complex indicates the presence of an Anaplasma phagosite film polypeptide or an Anaplasma platis polypeptide in the test sample. 15. The one or more antibodies are monoclonal antibodies, polyclonal antibodies, Fab fragments, Fab ′ fragments, Fab′-SH fragments, F (ab ′) ₂ fragments, Fv fragments, or single chain antibodies. the method of.   The composition of claim 1 further comprising a pharmaceutically acceptable or veterinary acceptable carrier.   20. The composition of claim 19, further comprising an adjuvant.   21. A method of treating or ameliorating Anaplasma platis infection, Anaplasma phagosite film infection, or both in a mammalian subject, wherein the mammalian subject is administered a therapeutically effective amount of the composition of Claim 19. The above method, comprising:   20. A method for raising an immune response in a mammal, comprising administering to said mammal an immunologically effective amount of the composition of claim 19.

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